Download Aminoacylated tmRNA from Escherichia coli interacts with

Aminoacylated tmRNA from Escherichia coli interacts
with prokaryotic elongation factor Tu.
Joëlle Rudinger-Thirion, Richard Giegé, Brice Felden
To cite this version:
Joëlle Rudinger-Thirion, Richard Giegé, Brice Felden. Aminoacylated tmRNA from Escherichia
coli interacts with prokaryotic elongation factor Tu.. RNA, Cold Spring Harbor Laboratory
Press, 1999, 5 (8), pp.989-92.
HAL Id: inserm-00718433
http://www.hal.inserm.fr/inserm-00718433
Submitted on 17 Jul 2012
HAL is a multi-disciplinary open access
archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.
RNA (1999), 5:989–992+ Cambridge University Press+ Printed in the USA+
Copyright © 1999 RNA Society+
LETTER TO THE EDITOR
Aminoacylated tmRNA from Escherichia coli
interacts with prokaryotic elongation factor Tu
JOËLLE RUDINGER-THIRION,1 RICHARD GIEGÉ,1 and BRICE FELDEN 2
1
Unité Propre de Recherche 9002 du Centre National de la Recherche Scientifique,
Institut de Biologie Moléculaire et Cellulaire, F-67084 Strasbourg, France
2
Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112-5330, USA
Keywords: 10Sa RNA; alanylation; minisubstrate; ternary complex; trans -translation
alanyl-tRNA synthetase (Komine et al+, 1994; Ushida
et al+, 1994), and tRNA modifying enzymes (Felden,
unpubl+ results)+ What about the other tRNA specific
proteins involved in translation?
E. coli tmRNA is found associated with 70S ribosomes in vivo, at about one molecule per 10 ribosomes
(Ushida et al+, 1994; Komine et al+, 1996)+ How tmRNA
enters the ribosomal A-site remains unknown+ It could
either form a ternary complex after alanylation with
elongation factor Tu (EF-TU) and guanosine-59-triphosphate (GTP), or use another specific pathway+ Biochemical studies (Rudinger et al+, 1994) as well as the X-ray
structures of two ternary complexes (Nissen et al+, 1995,
1999) indicate that tRNAs interact with prokaryotic EFTu-GTP via the aminoacylated 39 end, the phosphorylated 59 end, and the first 10 bp of the acceptor branch+
Crystal structures also show that EF-Tu contacts mainly
the ribose–phosphate backbone of the acceptor branch,
thus accommodating sequence variability within elongator tRNAs+ E. coli tmRNA contains a 7-bp acceptor
stem followed by a 5-bp T-stem, a 7-nt T-loop, and a
classical XCCA single-stranded 39 end (Fig+ 1B), as in
canonical elongator tRNAs+ A sequence comparison
between E. coli tmRNA and tRNAAla acceptor branches
indicates that 6 out of the first 10 bp of tmRNA are
identical to that of tRNAAla (Fig+ 1)+ This suggests that
alanylated tmRNA could be recognized by activated
EF-Tu+
The aim of this study was to test the above assumption and to discover whether aminoacylated tmRNA
forms a ternary complex with Thermus thermophilus
EF-Tu and GTP+ The choice of the thermophilic protein
was dictated by the available crystal structures of ternary complexes comprising this factor, considered to
be canonical models for any ternary complex (Nissen
et al+, 1995, 1999)+ Hydrolysis-protection assays were
used to monitor the ternary complex formation+ They
are based on the fact that the activated factor, when
Eubacterial tmRNAs (10Sa RNAs) are unique because
they function, at least in Escherichia coli, both as tRNA
and mRNA (for a review, see Muto et al+, 1998)+ These
;360 6 40-nt-long RNAs are charged with alanine at
their 39 ends by alanyl-tRNA synthetases or AlaRS
(Komine et al+, 1994; Ushida et al+, 1994)+ Alanylation
occurs thanks to the presence of the equivalent of the
G3-U70 pair, the major identity element for the alanylation of canonical tRNAs (Hou & Schimmel, 1988;
McClain & Foss, 1988)+ Bacterial tmRNAs also have a
short reading frame coding for 9 to 27 amino acids,
depending on the species+ E. coli tmRNA mediates recycling of ribosomes stalled at the end of terminatorless mRNAs, via a trans -translation process (Tu et al+,
1995; Keiler et al+, 1996; Himeno et al+, 1997; Withey &
Friedman, 1999)+ In E. coli, this amino acid tag is cotranslationally added to polypeptides synthesized from
mRNAs lacking a termination codon, and the added
11-amino-acid C-terminal tag makes the protein a target for specific proteolysis (Keiler et al+, 1996)+
Structural analyses based on phylogenetic (Felden
et al+, 1996; Williams & Bartel, 1996) and probing
(Felden et al+, 1997; Hickerson et al+, 1998) data have
led to a compact secondary structure model of E. coli
tmRNA+ These molecules have structural similarities
with canonical tRNAs, especially with tRNA acceptor
branches (Fig+ 1)+ Further E. coli tmRNA contains two
modified nucleosides, 5-methyluridine and pseudouridine, located in the T-loop mimic of the molecule (Felden
et al+, 1998)+ Its mimicry with tRNA occurs also at the
functional level, as it was already shown that tmRNA
interacts with certain tRNA specific proteins such as
RNase P (Komine et al+, 1994), RNase III (Srivastava
et al+, 1990; Makarov & Apirion, 1992), AlaRS, the
Reprint requests to: Brice Felden, Department of Human Genetics, University of Utah, 15N 2030E Room 6250, Salt Lake City, Utah
84112-5330, USA; e-mail: bfelden@genetics+utah+edu+
989
J. Rudinger-Thirion et al.
990
FIGURE 1. Comparison of tRNAAla (A) and tmRNA (B) secondary structures from E. coli+ The acceptor and acceptor-like
branches of the two RNAs are in red+ Part of the tRNA that interacts with prokaryotic elongation factor Tu is boxed in blue+
Nucleotides of tRNAAla are numbered according to Sprinzl et al+ (1998)+ For E. coli tmRNA, the six RNA helices (H1–H6) and
four RNA pseudoknots (PK1–PK4) are indicated+ These two RNAs are chargeable at their 39 ends with alanine as indicated+
bound to aminoacyl-RNAs, protects the labile amino
acid ester bond from spontaneous base-catalyzed hydrolysis+ Results are displayed in Figure 2+ As shown in
panels A and B, T. thermophilus EF-Tu protects the
deacylation of alanylated wild-type E. coli tmRNA similarly or even better than for alanylated tRNAAla + The
calculated half-life (t1/2 ) of the Ala-tmRNA ester bond is
17 min without EF-Tu-GTP and over 6 h in its presence
(Table 1)+ Similar results are obtained with the alanylated tmRNA transcript deprived of posttranscriptional
modifications (Fig+ 2C), with a t1/2 of 21 min of the ester
bound without EF-Tu and of about 5 h when the factor
is present (Table 1)+ Altogether, these results support
an interaction between charged tmRNA and EF-TuGTP comparable to that occurring with tRNAAla + These
measurements also show that the two modified bases
in E. coli tmRNA are dispensable for the interaction,
and that the additional sequence and intricate structure
of tmRNA compared to canonical tRNAs do not prevent
its recognition by EF-Tu-GTP+
Given these results, a charged minihelix derived from
the tmRNA acceptor branch may also interact with EFTu-GTP, provided the remaining part of tmRNA structure does not contain any positive signals indispensable
for the recognition+ The 6 first bp of the acceptor stem
in both E. coli tRNAAla and tmRNA are identical, so any
putative negative signals could only be located within
the 4 following bp+ Thus, minihelices of 35 nt recapitulating the acceptor branch of tRNAAla and of tmRNA
were prepared by in vitro transcription+ They derive
from the exact sequence of the two RNAs (Fig+ 1), and
were obtained after connection of nucleotides A7 to A49
in E. coli tRNAAla and G7 to G336 in E. coli tmRNA+ The
two alanylated RNA minisubstrates are both protected
against deacylation in the presence of EF-Tu-GTP
(t1/2 5 114 min for minihelix Ala and t1/2 5 172 min for
minihelix tmRNA ) albeit about twofold less than the fulllength RNAs (Table 1 and Fig+ 2D,E)+ This behavior is
reminiscent of that observed with aspartylated minihelices that also display increased half-lives as compared
to the whole molecule (Rudinger et al+, 1994)+ These
data suggest that in both tRNA and tmRNA, the connection of the acceptor branch to the remaining RNA
structure introduces similar structural constraints that
allow EF-Tu to bind+
The data presented here demonstrate the potential
of tmRNA to be recognized by EF-Tu, and therefore
likely rules out the requirement of a specialized factor
for its delivery to the ribosome+ This contrasts with other
tRNAs bearing specialized functions in translation that
make use of specific strategies for their delivery to the
ribosome+ Initiator tRNAs are brought to the ribosomal
P-site by an initiation factor (Gualerzi & Pon, 1990) and
selenocysteine tRNAs to the A-site by the SELB factor
(Forchhammer et al+, 1989, 1990)+ In these two cases,
alternate strategies have been selected to preclude an
interaction with EF-Tu-GTP+ A C1-A72 mismatch in prokaryotic tRNAMet (Seong & RajBhandary, 1987; Seong
et al+, 1989) and an antideterminant box at the 8–10-bp
positions in E. coli tRNASec (Rudinger et al+, 1996) are
the negative elements that preclude these tRNAs from
being recognized by EF-Tu+ To fulfill their “ribosomal
gymnastic” in all eubacteria, with the exception of
the alpha-proteobacteria (note in the tmRNA Website,
Interaction of tmRNA with EF-Tu
991
TABLE 1+ Times required to hydrolyze 50% of the labile alanyl-ester
bound (t1/2 ) for the five RNAs in the presence and in the absence
of EF-Tu-GTP+ The values were deduced from the corresponding
hydrolysis rates (Fig+ 2)+
Half-lives of ala-RNAs (min)
RNAs
tRNAAla (transcript)
tmRNA (overproduced)
tmRNA (transcript)
minihelix Ala
minihelix tmRNA
2EF-Tu
1EF-Tu
22
17
21
17
20
294
.360
295
114
172
Williams, 1999; Felden et al+, 1999), a tentative speculation is that tmRNAs arose from smaller functional
RNA modules+ These preexisting RNAs could have
evolved from canonical tRNAAla (acceptor branch),
mRNAs, and from a stretch of pseudoknots that are
commonly found in noncoding RNAs+ The tRNA-derived
module has allowed tmRNAs to be recognized by EF-Tu,
and this may have been sustained as an indispensable
prerequisite for tmRNAs to be active in trans -translation+
mophilus HB8 (Limmer et al+, 1992) were purified from
overproducing strains as described+ Oligonucleotides were
from NAPS GmbH (Göttingen, Germany), radioactive L-[C 14 ]
alanine (150 mCi/mmol) from Amersham (Les Ulis, France),
restriction enzyme Bst N1 from New England Biolabs (Beverly, Massachusetts), and phosphoenolpyruvate, pyruvate kinase, and the ribonucleotides from Boehringer (Mannheim,
Germany)+
The overexpressed (.100-fold) E. coli tmRNA (Felden
et al+, 1997) and the corresponding T7 transcript (Felden
et al+, 1998) were produced and purified by established procedures+ The tRNAAla transcript was prepared according to
Frugier et al+ (1993)+ Minihelices derived from E. coli tRNAAla
and from E. coli tmRNA sequences were obtained by in vitro
transcription of single-stranded DNA templates as described
for aspartate minihelices (Frugier et al+, 1994)+
Alanylation of RNAs was performed in 100 mM Tris-HCl,
pH 8+0, 10 mM KCl, 5 mM MgCl2 , 2 mM ATP, 10 mM dithioerythritol, 30 mM L-[C 14 ] alanine, and 2+8 mM AlaRS for
45 min at 37 8C+ All reactions were stopped by phenol extraction of the enzyme in the presence of sodium acetate at
pH 4+5+ The RNAs were ethanol precipitated, washed, and
dried+ Prior to that treatment, an aliquot of each sample was
spotted on a 3MM Whatman filter and 5% trichloroacetic acid
precipitated to measure the amount of alanylated RNA+
Hydrolysis-protection assays were conducted essentially
as described (Pingould & Urbanke, 1980; Rudinger et al+,
1994)+ For each experiment, ;50 pmol of the aminoacylated
transcripts (taking into account their alanylation level) were
dissolved in 50 mM Tris-HCl, pH 7+5, 50 mM KCl, and 10 mM
MgCl2 and incubated with a 5- and 10-fold excess of EF-TuGTP in a final volume of 100 mL+ Controls were carried out in
parallel with EF-Tu replaced by 50 mM Tris-HCl, pH 7+5,
50 mM KCl, and 10 mM MgCl2 + After formation of the ternary
complex “EF-Tu-GTP-alanylated RNA” (10 min incubation on
ice), samples were further incubated at 37 8C to allow hydrolysis of the chemically unstable ester bond to proceed+ Aliquots (20 mL) were pulled out at 0, 30, 60, and 90 min and
spotted onto 3MM Whatman filters+ After trichloroacetic acid
precipitation, the residual radioactivity was measured by liquid scintillation counting+ Each set of experiments was repeated twice+
MATERIALS AND METHODS
ACKNOWLEDGMENTS
AlaRS from E. coli (Hill & Schimmel, 1989), T7 RNA polymerase (Becker et al+, 1996), and EF-Tu-GDP from T. ther-
We are indebted to M+ Sprinzl (Bayreuth) for the generous gift
of a clone of T. thermophilus EF-Tu and a sample of the
FIGURE 2. Rates of hydrolysis of the 39-terminal alanine residue
from five L-[C 14 ]Ala-RNAs in the presence (d ) and the absence (Ä)
of Thermus thermophilus EF-Tu-GTP+ The concentration of labeled
Ala-RNA is 0+5 mM and that of EF-Tu-GTP is 2+5 mM+ A: alanylated
E. coli tRNAAla transcript; B: overproduced tmRNA; C: tmRNA transcript; D: minihelix Ala ; and E: minihelix tmRNA +
992
purified factor+ We are also grateful to M+ Frugier for purification of E. coli AlaRS and for providing the strain containing
the tRNAAla gene+ This work was partially supported by the
European Commission in the frame of the Biotechnology Program (BIO-98-0189), the Centre National de la Recherche
Scientifique, Université Louis Pasteur (Strasbourg)+ We are
deeply grateful for the support of Prof+ R+F+ Gesteland and Dr+
J+F+ Atkins+
Manuscript accepted without revision May 27, 1999
REFERENCES
Becker HD, Giegé R, Kern D+ 1996+ Identity of prokaryotic and eukaryotic tRNAAsp for aminoacylation by aspartyl-tRNA synthetase
from Thermus thermophilus+ Biochemistry 35 :7447–7458+
Felden B, Himeno H, Muto A, Atkins JF, Gesteland RF+ 1996+ Structural organization of Escherichia coli tmRNA+ Biochimie 78 :979–
983+
Felden B, Himeno H, Muto A, McCutcheon JP, Atkins JF, Gesteland
RF+ 1997+ Probing the structure of the Escherichia coli 10Sa RNA
(tmRNA)+ RNA 3 :89–103+
Felden B, Hanawa K, Atkins JF, Himeno H, Muto A, Gesteland RF,
McCloskey JA, Crain PF+ 1998+ Presence and location of modified nucleotides in Escherichia coli tmRNA: Structural mimicry
with tRNA acceptor branches+ EMBO J 17 :3188–3196+
Felden B, Gesteland RF, Atkins JF+ 1999+ Eubacterial tmRNAs: Everywhere except the Alpha-Proteobacteria? Biochim Biophys Acta
1446 :145–148+
Forchhammer K, Leinfelder W, Böck A+ 1989+ Identification of a novel
translation factor necessary for the incorporation of selenocysteine into protein+ Nature 342 :453– 456+
Forchhammer K, Ruecknagel K-P, Böck A+ 1990+ Purification and
biochemical characterization of SELB, a translation factor involved in selenoprotein synthesis+ J Biol Chem 265 :9346–9350+
Frugier M, Florentz C, Schimmel P, Giegé R+ 1993+ Triple aminoacylation specificity of a chimerized transfer RNA+ Biochemistry
32 :14053–14061+
Frugier M, Florentz C, Giegé R+ 1994+ Efficient aminoacylation of
resected RNA helices by class II aspartyl-tRNA synthetases+ Biochemistry 33 :9912–9921+
Gualerzi CO, Pon CL+ 1990+ Initiation of mRNA translation in prokaryotes+ Biochemistry 29 :5881–5889+
Hickerson RP, Watkins-Sims CD, Burrows CJ, Atkins JF, Gesteland
RF, Felden B+ 1998+ A nickel complex cleaves uridines in folded
RNA structures: Application to E. coli tmRNA and related engineered molecules+ J Mol Biol 279 :577–587+
Hill K, Schimmel P+ 1989+ Evidence that the 39-end of a transfer RNA
binds to a site in the adenylate synthesis domain of an aminoacyl-tRNA synthetase+ Biochemistry 28 :2577–2586+
Himeno H, Sato M, Tadaki T, Fukushima M, Ushida C, Muto A+ 1997+
In vitro trans translation mediated by alanine-charged 10Sa RNA+
J Mol Biol 268 :803–808+
Hou Y-M, Schimmel P+ 1988+ A simple structural feature is a major
determinant of the identity of a transfer RNA+ Nature 333 :140–
145+
Keiler KC, Waller PRH, Sauer RT+ 1996+ Role of a peptide tagging
system in degradation of proteins synthesized from damaged
messenger RNA+ Science 271:990–993+
J. Rudinger-Thirion et al.
Komine Y, Kitabatake M, Yokogawa T, Nishikawa K+ 1994+ A tRNA-like
structure is present in 10Sa RNA, a small stable RNA from Escherichia coli+ Proc Natl Acad Sci USA 91:9223–9227+
Komine Y, Kitabatake M, Inokuchi H+ 1996+ 10Sa RNA is associated
with 70S ribosome particles in Escherichia coli+ J Biochem (Tokyo)
119 :463– 467+
Limmer S, Reiser COA, Schirmer NK, Grillenbeck N, Sprinzl M+ 1992+
Nucleotide binding and GTP hydrolysis by elongation factor Tu
from Thermus thermophilus as monitored by proton NMR+ Biochemistry 31:2970–2977+
Makarov EM, Apirion D+ 1992+ 10Sa RNA: Processing by and inhibition of RNase III+ Biochem Int 26 :1115–1124+
McClain WH, Foss K+ 1988+ Changing the identity of a tRNA by
introducing a G-U wobble pair near the 39 acceptor end+ Science
240 :793–796+
Muto A, Ushida C, Himeno H+ 1998+ A bacterial RNA that functions as
both a tRNA and an mRNA+ Trends Biochem Sci 23 :25–29+
Nissen P, Kjeldgaard M, Thirup S, Polekhina G, Reshetnikova L,
Clark BFC, Nyborg J+ 1995+ Crystal structure of the ternary
complex of Phe-tRNAPhe , EF-Tu, and a GTP analog+ Science
270 :1464–1472+
Nissen P, Thirup S, Kjeldgaard M, Nyborg J+ 1999+ The crystal structure of Cys-tRNACys -EF-Tu-GDPNP reveals general and specific
features in the ternary complex and in tRNA+ Structure 7 :143–
156+
Pingould A, Urbanke C+ 1980+ Aminoacyl transfer ribonucleic acid
binding site of the bacterial elongation factor Tu+ Biochemistry
19 :2108–2112+
Rudinger J, Blechschmidt B, Ribeiro S, Sprinzl M+ 1994+ Minimalist
aminoacylated RNAs as efficient substrates for elongation factor
Tu+ Biochemistry 33 :5682–5688+
Rudinger J, Hillenbrandt R, Sprinzl M, Giegé R+ 1996+ Antideterminants present in minihelix Sec hinder its recognition by prokaryotic
elongation factor Tu+ EMBO J 15 :650– 657+
Seong BL, RajBhandary UL+ 1987+ Mutants of Escherichia coli formylmethionine tRNA: A single base change enables initiator tRNA to
act as an elongator in vitro. Proc Natl Acad Sci USA 84 :8859–
8863+
Seong BL, Lee CP, RajBhandary UL+ 1989+ Suppression of amber
codons in vivo as evidence that mutants derived from Escherichia
coli initiator tRNA can act as the step of elongation in protein
synthesis+ J Biol Chem 246 :6504–6508+
Sprinzl M, Horn C, Brown M, Ioudovitch A, Steinberg S+ 1998+ Compilation of tRNA sequences and sequences of tRNA genes+ Nucleic Acids Res 26 :148–153+
Srivastava RK, Miczak A, Apirion D+ 1990+ Maturation of precursor
10Sa RNA in Escherichia coli is a two-step process: The first
reaction is catalyzed by RNase III in presence of Mn 21 + Biochimie
72 :791–802+
Tu GF, Reid GE, Zhang JG, Moritz RL, Simpson RJ+ 1995+ C-terminal
extension of truncated recombinant proteins in Escherichia coli
with a 10Sa RNA decapeptide+ J Biol Chem 270 :9322–9326+
Ushida C, Himeno H, Watanabe T, Muto A+ 1994+ tRNA-like structures in 10Sa RNAs of Mycoplasma capricolum and Bacillus subtilis+ Nucleic Acids Res 22 :3392–3396+
Williams KP, Bartel DP+ 1996+ Phylogenetic analysis of tmRNA secondary structure+ RNA 2 :1306–1310+
Williams KP+ 1999+ The tmRNA Website+ Nucleic Acids Res 27 :165–
166+
Withey J, Friedman D+ 1999+ Analysis of the role of trans -translation
in the requirement of tmRNA for imm P22 growth in Escherichia
coli+ J Bacteriol 181:2148–2157+